Mono- and Di-Peg IL-10 Production; and Uses

ABSTRACT

Provided are methods of producing mono- and di-pegylated IL-10.

FIELD OF THE INVENTION

The present invention encompasses mono-pegylated (PEG) and di-PEG IL-10compositions and methods of use.

BACKGROUND OF THE INVENTION

The cytokine interleukin-10 (IL-10) is a dimer that becomes biologicallyinactive upon disruption of the non-covalent interactions connecting itstwo monomer subunits. IL-10 was first identified as a product of thetype 2 helper T cell and later shown to be produced by other cell typesincluding B cells and macrophages. It also inhibits the synthesis ofseveral cytokines produced from type 1 helper T cells, such asγ-interferon, IL-2, and tumor necrosis factor-α (TNF-α). The ability ofIL-10 to inhibit cell-mediated immune response modulators and suppressantigen-presenting cell-dependent T cell responses demonstrates IL-10has immunosuppressive properties. This cytokine also inhibitsmonocyte/macrophage production of other cytokines such as IL-1, IL-6,IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF),granulocyte colony-stimulating factor (G-CSF), and TNF-α. As a result ofits pleiotropic activity, IL-10 is under investigation for numerousclinical applications, such as for treating inflammatory conditions,bacterial sepsis, enterotoxin-induced lethal shock, and autoimmunediseases, e.g., rheumatoid arthritis, allograft rejection and diabetes.

Cancers and tumors can be controlled or eradicated by the immune system.The immune system includes several types of lymphoid and myeloid cells,e.g., monocytes, macrophages, dendritic cells (DCs), eosinophils, Tcells, B cells, and neutrophils. These lymphoid and myeloid cellsproduce secreted signaling proteins known as cytokines. The cytokinesinclude, e.g., interleukin-10 (IL-10), interferon-gamma (IFNγ), IL-12,and IL-23. Immune response includes inflammation, i.e., the accumulationof immune cells systemically or in a particular location of the body. Inresponse to an infective agent or foreign substance, immune cellssecrete cytokines which, in turn, modulate immune cell proliferation,development, differentiation, or migration. Excessive immune responsecan produce pathological consequences, such as autoimmune disorders,whereas impaired immune response may result in cancer. Anti-tumorresponse by the immune system includes innate immunity, e.g., asmediated by macrophages, NK cells, and neutrophils, and adaptiveimmunity, e.g., as mediated by antigen presenting cells (APCs), T cells,and B cells (see, e.g., Abbas, et al. (eds.) (2000) Cellular andMolecular Immunology, W.B. Saunders Co., Philadelphia, Pa.; Oppenheimand Feldmann (eds.) (2001) Cytokine Reference, Academic Press, SanDiego, Calif.; von Andrian and Mackay (2000) New Engl. J. Med.343:1020-1034; Davidson and Diamond (2001) New Engl. J. Med.345:340-350).

Methods of modulating immune response have been used in the treatment ofcancers, e.g., melanoma. These methods include treatment either withcytokines such as IL-2, IL-10, IL-12, tumor necrosis factor-alpha(TNFalpha), IFNγ, granulocyte macrophage-colony stimulating factor(GM-CSF), and transforming growth factor (TGF), or with cytokineantagonists (e.g., antibodies). Interleukin-10 was first characterizedas a cytokine synthesis inhibitory factor (CSIF; see, e.g., Fiorentino,et al (1989) J. Exp. Med. 170:2081-2095). IL-10 is a pleiotropiccytokine produced by T cells, B cells, monocytes, that can function asboth an immunosuppressant and immunostimulant (see, e.g., Groux, et al.(1998) J. Immunol. 160:3188-3193; and Hagenbaugh, et al. (1997) J. Exp.Med. 185:2101-2110).

Animal models suggest that IL-10 can induce NK-cell activation andfacilitate target-cell destruction in a dose-dependent manner (see,e.g., Zheng, et al. (1996) J. Exp. Med. 184:579-584; Kundu, et al.(1996) J. Natl. Cancer Inst. 88:536-541). Further studies indicate thatthe presence of IL-10 in the tumor microenvironment correlates withbetter patient survival (see, e.g., Lu, et al. (2004) J. Clin. Oncol.22:4575-4583).

Because of its relatively short half life, IL-10 has been conjugated tovarious partners, including polyethylene glycol. Other cytokines havealso been pegylated, generally via monopegylation, e.g., PEG moleculesattached to a single residue on the cytokine protein. Unfortunately,monopegylation on one IL-10 subunit leads to a non-homogenous mix ofdipegylated, monopegylated and nonpegylated IL-10 molecules due tosubunit shuffling. Allowing a pegylation reaction to proceed tocompletion will also permit non-specific and multi-pegylated targetproteins, thus reducing the bioactivity of these proteins. Thus a needexists to more efficiently produce correctly pegylated IL-10 withgreater production yields. The present invention satisfies this need byproviding methods of producing a mixture of mono- and di-pegylatedIL-10.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the reaction kinetics of producing mono- and di-PEG-IL-10.

FIG. 2 shows the efficacy of various mono- and diPEG-IL-10 (murine)prototypes on implanted PDV6 squamous cell carcinomas.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery that a controlledreaction will produce a mix of selectively pegylated mono- anddi-PEG-IL-10 which in turn improves yield of final pegylated product andhas comparable efficacy to other PEG-IL-10 species.

The present invention provides a method of producing a mixture of mono-and di-pegylated IL-10, wherein at least one PEG molecule is covalentlyattached to at least one amino acid residue of at least one subunit ofIL-10, comprising: a) reacting 1 mg/ml to 12 mg/ml of IL-10 protein withan activated PEG-linker such that the IL-10 to PEG-linker ratio is 1:1to 1:7.7, in the presence of 0.75 mM to 35 mM of a reducing agent, at apH of about 5.0 to 7.4 and a temperature of 5° C. to 30° C., for 12-15hours; and b) purifying the mixture of mono- and di-pegylated IL-10. Incertain embodiments, the PEG-linker is selected from the groupconsisting of succinimidylcarbonate-PEG, PEG-butyraldehyde,PEG-pentaldehyde, PEG-amido-propionaldehyde,PEG-urethano-propioaldehyde, and PEG-propylaldehyde, the PEG-linker isfrom 5,000 daltons to 12,000 daltons, or the reducing agent is selectedfrom the group consisting of borohydride, sodium cyanoborohydride, amineborane, and picoline borane. In yet another embodiment, the mixture ofmono- and di-PEG is purified by chromatography selected from the groupconsisting of cation exchange, anion exchange, size exclusion, andhydrophobic interaction. Also encompassed is a pharmaceuticalcomposition comprising the mono- and di-PEG-IL-10 produced by thisreaction method and a pharmaceutically acceptable carrier.

The present invention encompasses a method of producing a mixture ofmono- and di-pegylated IL-10, wherein at least one PEG molecule iscovalently attached to at least one amino acid residue of at least onesubunit of IL-10, comprising a) reacting 7.5 mg/ml of IL-10 with anactivated PEG-linker such that the IL-10 to PEG-linker ratio is 1:3.5,in the presence of 25 mM of a reducing agent, at a pH of 6.3 and atemperature of 15° C., for 15 hours; and b) purifying the mixture ofmono- and di-pegylated IL-10. In certain embodiments, the PEG-linker isselected from the group consisting of succinimidylcarbonate-PEG,PEG-butyraldehyde, PEG-pentaldehyde, PEG-amido-propionaldehyde,PEG-urethano-propioaldehyde, and PEG-propylaldehyde. The molecular massof PEG comprising the PEG-linker is from 5,000 daltons to 20,000daltons, the reducing agent is selected from the group consisting ofborohydride, sodium cyanoborohydride, amine borane, and picoline boraneor the mixture of mono- and di-PEG is purified by chromatographyselected from the group consisting of cation exchange, anion exchange,size exclusion, and hydrophobic interaction. Also encompassed is apharmaceutical composition comprising the mono- and di-PEG-IL-10produced by this reaction method and a pharmaceutically acceptablecarrier.

DETAILED DESCRIPTION

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the,” include their corresponding pluralreferences unless the context clearly dictates otherwise. All referencescited herein are incorporated by reference to the same extent as if eachindividual publication, patent application, or patent, was specificallyand individually indicated to be incorporated by reference.

I. DEFINITIONS

“Activation,” “stimulation,” and “treatment,” as it applies to cells orto receptors, may have the same meaning, e.g., activation, stimulation,or treatment of a cell or receptor with a ligand, unless indicatedotherwise by the context or explicitly. “Ligand” encompasses natural andsynthetic ligands, e.g., cytokines, cytokine variants, analogues,muteins, and binding compositions derived from antibodies. “Ligand” alsoencompasses small molecules, e.g., peptide mimetics of cytokines andpeptide mimetics of antibodies. “Activation” can refer to cellactivation as regulated by internal mechanisms as well as by external orenvironmental factors. “Response,” e.g., of a cell, tissue, organ, ororganism, encompasses a change in biochemical or physiological behavior,e.g., concentration, density, adhesion, or migration within a biologicalcompartment, rate of gene expression, or state of differentiation, wherethe change is correlated with activation, stimulation, or treatment, orwith internal mechanisms such as genetic programming.

“Activity” of a molecule may describe or refer to the binding of themolecule to a ligand or to a receptor, to catalytic activity; to theability to stimulate gene expression or cell signaling, differentiation,or maturation; to antigenic activity, to the modulation of activities ofother molecules, and the like. “Activity” of a molecule may also referto activity in modulating or maintaining cell-to-cell interactions,e.g., adhesion, or activity in maintaining a structure of a cell, e.g.,cell membranes or cytoskeleton. “Activity” can also mean specificactivity, e.g., [catalytic activity]/[mg protein], or [immunologicalactivity]/[mg protein], concentration in a biological compartment, orthe like. “Proliferative activity” encompasses an activity thatpromotes, that is necessary for, or that is specifically associatedwith, e.g., normal cell division, as well as cancer, tumors, dysplasia,cell transformation, metastasis, and angiogenesis.

“Administration” and “treatment,” as it applies to an animal, human,experimental subject, cell, tissue, organ, or biological fluid, refersto contact of an exogenous pharmaceutical, therapeutic, diagnosticagent, compound, or composition to the animal, human, subject, cell,tissue, organ, or biological fluid. “Administration” and “treatment” canrefer, e.g., to therapeutic, placebo, pharmacokinetic, diagnostic,research, and experimental methods. “Treatment of a cell” encompassescontact of a reagent to the cell, as well as contact of a reagent to afluid, where the fluid is in contact with the cell. “Administration” and“treatment” also means in vitro and ex vivo treatments, e.g., of a cell,by a reagent, diagnostic, binding composition, or by another cell.“Treatment,” as it applies to a human, veterinary, or research subject,refers to therapeutic treatment, prophylactic or preventative measures,to research and diagnostic applications. “Treatment” as it applies to ahuman, veterinary, or research subject, or cell, tissue, or organ,encompasses contact of PEG-IL-10 to a human or animal subject, a cell,tissue, physiological compartment, or physiological fluid. “Treatment ofa cell” also encompasses situations where PEG-IL-10 contacts IL-10receptor (heterodimer of IL-10R1 and IL-10R2) e.g., in the fluid phaseor colloidal phase, as well as situations where an IL-10 agonist orantagonist contacts a fluid, e.g., where the fluid is in contact with acell or receptor, but where it has not been demonstrated that theagonist or antagonist directly contacts the cell or receptor.

“Cachexia” is a wasting syndrome involving loss of muscle (musclewasting) and fat, resulting from a disorder in metabolism. Cachexiaoccurs in various cancers (“cancer cachexia”), chronic pulmonaryobstructive disorder (COPD), advanced organ failure, and AIDS. Cancercachexia is characterized by, e.g., marked weight loss, anorexia,asthenia, and anemia. Anorexia is a disorder resulting from lack ofmotivation to eat, e.g., food aversion (see, e.g., MacDonald, et al.(2003) J. Am. Coll. Surg. 197:143-161; Rubin (2003) Proc. Natl. Acad.Sci. USA 100:5384-5389; Tisdale (2002) Nature Reviews Cancer 2:862-871;Argiles, et al. (2003) Drug Discovery Today 8:838-844; Lelli, et al.(2003) J. Chemother. 15:220-225; Argiles, et al. (2003) Curr. Opin.Clin. Nutr. Metab. Care 6:401-406).

“Conservatively modified variants of PEG-IL-10” applies to both aminoacid and nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences or, where the nucleic acid does not encode an amino acidsequence, to essentially identical nucleic acid sequences. Because ofthe degeneracy of the genetic code, a large number of functionallyidentical nucleic acids may encode any given protein.

As to amino acid sequences, one of skill will recognize that anindividual substitution to a nucleic acid, peptide, polypeptide, orprotein sequence which substitutes an amino acid or a small percentageof amino acids in the encoded sequence for a conserved amino acid is a“conservatively modified variant.” Conservative substitution tablesproviding functionally similar amino acids are well known in the art. Anexample of a conservative substitution is the exchange of an amino acidin one of the following groups for another amino acid of the same group(U.S. Pat. No. 5,767,063 issued to Lee, et al.; Kyte and Doolittle(1982) J. Mol. Biol. 157: 105-132):

(1) Hydrophobic: Norleucine, Ile, Val, Leu, Phe, Cys, or Met;

(2) Neutral hydrophilic: Cys, Ser, Thr;

(3) Acidic: Asp, Glu; (4) Basic: Asn, Gln, His, Lys, Arg;

(5) Residues that influence chain orientation: Gly, Pro;

(6) Aromatic: Tip, Tyr, Phe;

(7) Small amino acids: Gly, Ala, Ser.

“Effective amount” encompasses an amount sufficient to ameliorate orprevent a symptom or sign of the medical condition. Effective amountalso means an amount sufficient to allow or facilitate diagnosis. Aneffective amount for a particular patient or veterinary subject may varydepending on factors such as the condition being treated, the overallhealth of the patient, the method route and dose of administration andthe severity of side effects (see, e.g., U.S. Pat. No. 5,888,530 issuedto Netti, et al.). An effective amount can be the maximal dose or dosingprotocol that avoids significant side effects or toxic effects. Theeffect will result in an improvement of a diagnostic measure orparameter by at least 5%, usually by at least 10%, more usually at least20%, most usually at least 30%, preferably at least 40%, more preferablyat least 50%, most preferably at least 60%, ideally at least 70%, moreideally at least 80%, and most ideally at least 90%, where 100% isdefined as the diagnostic parameter shown by a normal subject (see,e.g., Maynard, et al. (1996) A Handbook of SOPs for Good ClinicalPractice, Interpharm Press, Boca Raton, Fla.; Dent (2001) GoodLaboratory and Good Clinical Practice, Urch Publ., London, UK). Aneffective amount of PEG-IL-10 would be an amount sufficient to reduce atumor volume, inhibit tumor growth, prevent metastasis, or increase CD8+T cell infiltration in to the tumor site.

“Exogenous” refers to substances that are produced outside an organism,cell, or human body, depending on the context. “Endogenous” refers tosubstances that are produced within a cell, organism, or human body,depending on the context.

“Immune condition” or “immune disorder” encompasses, e.g., pathologicalinflammation, an inflammatory disorder, and an autoimmune disorder ordisease. “Immune condition” also refers to infections, persistentinfections, and proliferative conditions, such as cancer, tumors, andangiogenesis, including infections, tumors, and cancers that resistirradiation by the immune system. “Cancerous condition” includes, e.g.,cancer, cancer cells, tumors, angiogenesis, and precancerous conditionssuch as dysplasia.

“Inhibitors” and “antagonists” or “activators” and “agonists” refer toinhibitory or activating molecules, respectively, e.g., for theactivation of, e.g., a ligand, receptor, cofactor, gene, cell, tissue,or organ. A modulator of, e.g., a gene, a receptor, a ligand, or a cell,is a molecule that alters an activity of the gene, receptor, ligand, orcell, where activity can be activated, inhibited, or altered in itsregulatory properties. The modulator may act alone, or it may use acofactor, e.g., a protein, metal ion, or small molecule Inhibitors arecompounds that decrease, block, prevent, delay activation, inactivate,desensitize, or down regulate, e.g., a gene, protein, ligand, receptor,or cell. Activators are compounds that increase, activate, facilitate,enhance activation, sensitize, or up regulate, e.g., a gene, protein,ligand, receptor, or cell. An inhibitor may also be defined as acomposition that reduces, blocks, or inactivates a constitutiveactivity. An “agonist” is a compound that interacts with a target tocause or promote an increase in the activation of the target. An“antagonist” is a compound that opposes the actions of an agonist. Anantagonist prevents, reduces, inhibits, or neutralizes the activity ofan agonist. An antagonist can also prevent, inhibit, or reduceconstitutive activity of a target, e.g., a target receptor, even wherethere is no identified agonist.

To examine the extent of inhibition, for example, samples or assayscomprising a given, e.g., protein, gene, cell, or organism, are treatedwith a potential activator or inhibitor and are compared to controlsamples without the inhibitor. Control samples, i.e., not treated withantagonist, are assigned a relative activity value of 100% Inhibition isachieved when the activity value relative to the control is about 90% orless, typically 85% or less, more typically 80% or less, most typically75% or less, generally 70% or less, more generally 65% or less, mostgenerally 60% or less, typically 55% or less, usually 50% or less, moreusually 45% or less, most usually 40% or less, preferably 35% or less,more preferably 30% or less, still more preferably 25% or less, and mostpreferably less than 25%. Activation is achieved when the activity valuerelative to the control is about 110%, generally at least 120%, moregenerally at least 140%, more generally at least 160%, often at least180%, more often at least 2-fold, most often at least 2.5-fold, usuallyat least 5-fold, more usually at least 10-fold, preferably at least20-fold, more preferably at least 40-fold, and most preferably over40-fold higher.

Endpoints in activation or inhibition can be monitored as follows.Activation, inhibition, and response to treatment, e.g., of a cell,physiological fluid, tissue, organ, and animal or human subject, can bemonitored by an endpoint. The endpoint may comprise a predeterminedquantity or percentage of, e.g., an indicia of inflammation,oncogenicity, or cell degranulation or secretion, such as the release ofa cytokine, toxic oxygen, or a protease. The endpoint may comprise,e.g., a predetermined quantity of ion flux or transport; cell migration;cell adhesion; cell proliferation; potential for metastasis; celldifferentiation; and change in phenotype, e.g., change in expression ofgene relating to inflammation, apoptosis, transformation, cell cycle, ormetastasis (see, e.g., Knight (2000) Ann. Clin. Lab. Sci. 30:145-158;Hood and Cheresh (2002) Nature Rev. Cancer 2:91-100; Timme, et al.(2003) Curr. Drug Targets 4:251-261; Robbins and Itzkowitz (2002) Med.Clin. North Am. 86:1467-1495; Grady and Markowitz (2002) Annu. Rev.Genomics Hum. Genet. 3:101-128; Bauer, et al. (2001) Glia 36:235-243;Stanimirovic and Satoh (2000) Brain Pathol. 10:113-126).

An endpoint of inhibition is generally 75% of the control or less,preferably 50% of the control or less, more preferably 25% of thecontrol or less, and most preferably 10% of the control or less.Generally, an endpoint of activation is at least 150% the control,preferably at least two times the control, more preferably at least fourtimes the control, and most preferably at least 10 times the control.

A composition that is “labeled” is detectable, either directly orindirectly, by spectroscopic, photochemical, biochemical,immunochemical, isotopic, or chemical methods. For example, usefullabels include ³²P, ³³P, ³⁵S, ¹⁴C, ³H, ¹²⁵I, stable isotopes,fluorescent dyes, electron-dense reagents, substrates, epitope tags, orenzymes, e.g., as used in enzyme-linked immunoassays, or fluorettes(see, e.g., Rozinov and Nolan (1998) Chem. Biol. 5:713-728).

“Ligand” refers, e.g., to a small molecule, peptide, polypeptide, andmembrane associated or membrane-bound molecule, or complex thereof, thatcan act as an agonist or antagonist of a receptor. “Ligand” alsoencompasses an agent that is not an agonist or antagonist, but that canbind to the receptor without significantly influencing its biologicalproperties, e.g., signaling or adhesion. Moreover, “ligand” includes amembrane-bound ligand that has been changed, e.g., by chemical orrecombinant methods, to a soluble version of the membrane-bound ligand.By convention, where a ligand is membrane-bound on a first cell, thereceptor usually occurs on a second cell. The second cell may have thesame or a different identity as the first cell. A ligand or receptor maybe entirely intracellular, that is, it may reside in the cytosol,nucleus, or some other intracellular compartment. The ligand or receptormay change its location, e.g., from an intracellular compartment to theouter face of the plasma membrane. The complex of a ligand and receptoris termed a “ligand receptor complex.” Where a ligand and receptor areinvolved in a signaling pathway, the ligand occurs at an upstreamposition and the receptor occurs at a downstream position of thesignaling pathway.

“Small molecules” are provided for the treatment of physiology anddisorders of tumors and cancers. “Small molecule” is defined as amolecule with a molecular weight that is less than 10 kD, typically lessthan 2 kD, and preferably less than 1 kD. Small molecules include, butare not limited to, inorganic molecules, organic molecules, organicmolecules containing an inorganic component, molecules comprising aradioactive atom, synthetic molecules, peptide mimetics, and antibodymimetics. As a therapeutic, a small molecule may be more permeable tocells, less susceptible to degradation, and less apt to elicit an immuneresponse than large molecules. Small molecules, such as peptide mimeticsof antibodies and cytokines, as well as small molecule toxins aredescribed (see, e.g., Casset, et al. (2003) Biochem. Biophys. Res.Commun. 307:198-205; Muyldermans (2001) J. Biotechnol. 74:277-302; Li(2000) Nat. Biotechnol. 18:1251-1256; Apostolopoulos, et al. (2002)Curr. Med. Chem. 9:411-420; Monfardini, et al. (2002) Curr. Pharm. Des.8:2185-2199; Domingues, et al. (1999) Nat. Struct. Biol. 6:652-656; Satoand Sone (2003) Biochem. J. 371:603-608; U.S. Pat. No. 6,326,482 issuedto Stewart, et al.).

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamime nitrogen mustardssuch as chiorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; antibiotics such asaclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL® Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (Taxotere™, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; Xeloda® Roche, Switzerland; ibandronate; CPT11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoic acid; esperamicins; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above. Alsoincluded in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogensincluding for example tamoxifen, raloxifene, aromatase inhibiting4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,onapristone, and toremifene (Fareston); and antiandrogens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin; andpharmaceutically acceptable salts, acids or derivatives of any of theabove

“Specifically” or “selectively” binds, when referring to aligand/receptor, antibody/antigen, or other binding pair, indicates abinding reaction which is determinative of the presence of the proteinin a heterogeneous population of proteins and other biologics. Thus,under designated conditions, a specified ligand binds to a particularreceptor and does not bind in a significant amount to other proteinspresent in the sample. The antibody, or binding composition derived fromthe antigen-binding site of an antibody, of the contemplated methodbinds to its antigen, or a variant or mutein thereof, with an affinitythat is at least two fold greater, preferably at least ten timesgreater, more preferably at least 20-times greater, and most preferablyat least 100-times greater than the affinity with any other antibody, orbinding composition derived thereof. In a preferred embodiment theantibody will have an affinity that is greater than about 10⁹liters/mol, as determined, e.g., by Scatchard analysis (Munsen, et al.(1980) Analyt. Biochem. 107:220-239).

“Interleukin-10” or “IL-10”, as used herein, whether conjugated to apolyethylene glycol, or in a non-conjugated form, is a proteincomprising two subunits nocovalently joined to form a homodimer. As usedherein, unless otherwise indicated “interleukin-10” and “IL-10” canrefer to human or mouse IL-10 (Genbank Accession Nos. NP_(—)000563;M37897; or U.S. Pat. No. 6,217,857) which are also referred to as“hIL-10” or “mIL-10”.

“Pegylated IL-10” or “PEG-IL-10” is an IL-10 molecule having one or morepolyethylene glycol molecules covalently attached to one or more thanone amino acid residue of the IL-10 protein via a linker, such that theattachment is stable. The terms “monopegylated IL-10” and“mono-PEG-IL-10”, mean that at least one polyethylene glycol molecule iscovalently attached to a single amino acid residue on one subunit of theIL-10 dimer via a linker. The terms “dipegylated IL-10” and“di-PEG-IL-10” mean that at least one PEG molecule is attached to asingle residue on each subunit of the IL-10 dimer via a linker. Theaverage molecular weight of the PEG moiety is preferably between about5,000 and about 50,000 daltons. The method or site of PEG attachment toIL-10 is not critical, but preferably the pegylation does not alter, oronly minimally alters, the activity of the biologically active molecule.Preferably, the increase in half-life is greater than any decrease inbiological activity. For PEG-IL-10, biological activity is typicallymeasured by assessing the levels of inflammatory cytokines (e.g., TNFα,IFNγ) in the serum of subjects challenged with a bacterial antigen(lipopolysaccharide, LPS) and treated with PEG-IL-10, as described inU.S. Pat. No. 7,052,686.

As used herein, “serum half-life”, abbreviated “t_(1/2)”, meanselimination half-life, i.e., the time at which the serum concentrationof an agent has reached one-half its initial or maximum value. The term“increased serum half-life” used herein in reference to a syntheticagent means that the synthetic agent is cleared at a slower rate thaneither the non-synthetic, endogenous agent or the recombinantly producedversion thereof.

II. GENERAL

The present invention provides methods of producing a mixture of mono-and di-PEG. Pegylated IL-10 has been shown to be more efficacious in atumor setting, see, e.g., US20080081031. The present invention providesa method to increase yields of pegylated IL-10 by purifying bothmonopegylated (at least one PEG molecule on one subunit of the IL-10homodimer) and dipegylated (at least one PEG molecule on each subunit ofthe IL-10 homodimer) IL-10.

III. POLYETHYLENE GLYCOL (“PEG”)

Polyethylene glycol (“PEG”) is a chemical moiety which has been used inthe preparation of therapeutic protein products. The verb “pegylate”means to attach at least one PEG molecule to another molecule, e.g. atherapeutic protein. For example Adagen, a pegylated formulation ofadenosine deaminase, is approved for treating severe combinedimmunodeficiency disease; pegylated superoxide dismutase has been inclinical trials for treating head injury; pegylated alpha interferon hasbeen tested in phase I clinical trials for treating hepatitis; pegylatedglucocerebrosidase and pegylated hemoglobin are reported to have been inpreclinical testing. The attachment of polyethylene glycol has beenshown to protect against proteolysis (see, e.g., Sada, et al., (1991) J.Fermentation Bioengineering 71:137-139).

In its most common form, PEG is a linear or branched polyetherterminated with hydroxyl groups and having the general structure:

HO—(CH₂CH₂O)_(n)—CH₂CH₂—OH

To couple PEG to a molecule (polypeptides, polysaccharides,polynucleotides, and small organic molecules) it is necessary toactivate the PEG by preparing a derivative of the PEG having afunctional group at one or both termini. The most common route for PEGconjugation of proteins has been to activate the PEG with functionalgroups suitable for reaction with lysine and N-terminal amino acidgroups. In particular, the most common reactive groups involved incoupling of PEG to polypeptides are the alpha or epsilon amino groups oflysine.

The reaction of a pegylation linker with a protein leads to theattachment of the PEG moiety predominantly at the following sites: thealpha amino group at the N-terminus of the protein, the epsilon aminogroup on the side chain of lysine residues, and the imidazole group onthe side chain of histidine residues. Since most recombinant proteinpossess a single alpha and a number of epsilon amino and imidazloegroups, numerous positional isomers can be generated depending on thelinker chemistry.

Two widely used first generation activated monomethoxy PEGs (mPEGs) weresuccinimdyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992)Biotehnol. Appl. Biochem 15:100-114; and Miron and Wilcheck (1993)Bioconjug. Chem. 4:568-569) and benzotriazole carbonate PEG (BTC-PEG;see, e.g., Dolence, et al. U.S. Pat. No. 5,650,234), which reactpreferentially with lysine residues to form a carbamate linkage, but arealso known to react with histidine and tyrosine residues. The linkage tohistidine residues on IFNα has been shown to be a hydrolyticallyunstable imidazolecarbamate linkage (see, e.g., Lee and McNemar, U.S.Pat. No. 5,985,263).

Second generation PEGylation technology has been designed to avoid theseunstable linkages as well as the lack of selectivity in residuereactivity. Use of a PEG-aldehyde linker targets a single site on theN-terminus of a polypeptide and/or protein subunit through reductiveamination. IL-10 may be pegylated using different types of linkers andpH to arrive at a various forms of a pegylated molecule (see, e.g., U.S.Pat. No. 5,252,714, U.S. Pat. No. 5,643,575, U.S. Pat. No. 5,919,455,U.S. Pat. No. 5,932,462, U.S. Pat. No. 5,985,263, U.S. Pat. No.7,052,686).

IV. BIOLOGICAL ACTIVITY OF PEG-IL-10

Human IL-10 induces rapid development of neutralizing antibodies whenadministered to immunocompetent mice. To avoid this type ofneutralization, subcutaneous administration of PEG-hIL-10 was given tomice deficient in B-cells, i.e., mice unable to mount an antibodyresponse. Well established syngeneic tumors in these immunodeficientmice were either significantly delayed in growth or rejected completelyby PEG-hIL-10. The tumor growth restriction or inhibition was dependenton both CD4 and CD8 T-cells. Upon depletion of CD8 cells, the inhibitoryeffect of PEG-hIL-10 was completely abrogated. Thus, PEG-hIL-10 inducesCD8 mediated cytotoxic responses.

Further analysis of tumor tissue showed that PEG-IL-10 increased theinfiltration of CD8+ T cells into the tumor at a level greater than thatof non-pegylated IL-10. The level of inflammatory cytokine expression bythe infiltrating CD8 cells was also higher with PEG-IL-10 treatment ascompared to non-pegylated IL-10 treatment. Treatment of tumor patientswith PEG-IL-10 should induce a significant antitumor response and confera significant therapeutic benefit (see, e.g.,

An IL-10 protein used in the present invention contains an amino acidsequence that shares an observed homology of at least 75%, morepreferably at least 85%, and most preferably at least 90% or more, e.g.,at least 95%, with the sequence of a mature IL-10 protein, i.e., lackingany leader sequences. See, e.g., U.S. Pat. No. 6,217,857. Amino acidsequence homology, or sequence identity, is determined by optimizingresidue matches and, if necessary, by introducing gaps as required.Homologous amino acid sequences are typically intended to includenatural allelic, polymorphic and interspecies variations in eachrespective sequence. Typical homologous proteins or peptides will havefrom 25-100% homology (if gaps can be introduced) to 50-100% homology(if conservative substitutions are included) with the amino acidsequence of the IL-10 polypeptide. See Needleham et al., J. Mol. Biol.48:443-453 (1970); Sankoff et al. in Time Warps, String Edits, andMacromolecules: The Theory and Practice of Sequence Comparison, 1983,Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif., and the University of WisconsinGenetics Computer Group, Madison, Wis.

The IL-10 moiety in the PEG-IL-10 conjugates can be glycosylated or maybe modified with unglycosylated muteins or other analogs, including theBCRF1 (Epstein Barr Virus viral IL-10) protein. Modifications ofsequences encoding IL-10 can be made using a variety of techniques,e.g., site-directed mutagenesis [Gillman et al., Gene 8:81-97 (1979);Roberts et al., Nature 328:731-734 (1987)], and can be evaluated byroutine screening in a suitable assay for IL-10 activity. Modified IL-10proteins, e.g., variants, can vary from the naturally-occurring sequenceat the-primary structure level. Such modifications can be made by aminoacid insertions, substitutions, deletions and fusions. IL-10 variantscan be prepared with various objectives in mind, including increasingserum half-life, reducing an immune response against the IL-10,facilitating purification or preparation, decreasing conversion of IL-10into its monomeric subunits, improving therapeutic efficacy, andlessening the severity or occurrence of side effects during therapeuticuse. The amino acid sequence variants are usually predetermined variantsnot found in nature, although others may be post-translational variants,e.g., glycosylated variants. Any variant of IL-10 can be used in thisinvention provided it retains a suitable level of IL-10 activity. In thetumor context, suitable IL-10 activity would be, e.g., CD8+ T cellinfiltrate into tumor sites, expression of inflammatory cytokines suchas IFNγ, IL-4, IL-6, IL-10, and RANK-L, from these infiltrating cells,increased levels of TNFα or IFNγ in biological samples,

IL-10 used in this invention can be derived from a mammal, e.g. human ormouse. Human IL-10 (hIL-10) is preferred for treatment of humans in needof IL-10 treatment. IL-10 used in this invention is preferably arecombinant IL-10. Methods describing the preparation of human and mouseIL-10 can be found in U.S. Pat. No. 5,231,012. Also included arenaturally occurring or conservatively substituted variants of human andmouse IL-10. In another embodiment of the present invention, IL-10 canbe of viral origin. The cloning and expression of a viral IL-10 fromEpstein Barr virus (BCRF1 protein) is disclosed in Moore et al., Science248:1230 (1990).

IL-10 can be obtained in a number of ways using standard techniquesknown in the art, e.g., isolated and purified from culture media ofactivated cells capable of secreting the protein (e.g., T-cells),chemically synthesized, or recombinant techniques, (see, e.g.,Merrifield, Science 233:341-47 (1986); Atherton et al., Solid PhasePeptide Synthesis, A Practical Approach, 1989, I.R.L. Press, Oxford;U.S. Pat. No. 5,231,012 which teaches methods for the production ofproteins having IL-10 activity, including recombinant and othersynthetic techniques). Preferably, IL-10 protein is obtained fromnucleic acids encoding the IL-10 polypeptide using recombinanttechniques. Recombinant human IL-10 is also commercially available,e.g., from PeproTech, Inc., Rocky Hill, N.J.

PEG-IL-10 can be made using techniques well known in the art.Polyethylene glycol (PEG) can be synthesized as described, e.g., inLundblad, R. L. et al. (1988) Chemical Reagents for Protein ModificationCRC Press, Inc., vol. 1, pp. 105-125. PEG can be conjugated to IL-10through use of a linker as described above. In certain embodiments, thePEG-IL-10 used in the invention is a mono-PEG-IL-10 in which one to ninePEG molecules are covalently attached via a linker to the alpha aminogroup of the amino acid residue at the N-terminus of one subunit of theIL-10 dimer.

IV. THERAPEUTIC COMPOSITIONS, METHODS

PEG-IL-10 can be formulated in a pharmaceutical composition comprising atherapeutically effective amount of the IL-10 and a pharmaceuticalcarrier. A “therapeutically effective amount” is an amount sufficient toprovide the desired therapeutic result. Preferably, such amount hasminimal negative side effects. The amount of PEG-IL-10 administered totreat a condition treatable with IL-10 is based on IL-10 activity of theconjugated protein, which can be determined by IL-10 activity assaysknown in the art. The therapeutically effective amount for a particularpatient in need of such treatment can be determined by consideringvarious factors, such as the condition treated, the overall health ofthe patient, method of administration, the severity of side-effects, andthe like. In the tumor context, suitable IL-10 activity would be, e.g.,CD8 T cell infiltrate into tumor sites, expression of inflammatorycytokines such as IFNγ, IL-4, IL-6, IL-10, and RANK-L, from theseinfiltrating cells, increased levels of TNFα or IFNγ in biologicalsamples.

The therapeutically effective amount of pegylated IL-10 can range fromabout 0.01 to about 100 μg protein per kg of body weight per day.Preferably, the amount of pegylated IL-10 ranges from about 0.1 to 20 μgprotein per kg of body weight per day, more preferably from about 0.5 to10 μg protein per kg of body weight per day, and most preferably fromabout 1 to 4 μg protein per kg of body weight per day. Less frequentadministration schedules can be employed using the PEG-IL-10 of theinvention since this conjugated form is longer acting than IL-10. Thepegylated IL-10 is formulated in purified form and substantially free ofaggregates and other proteins. Preferably, PEG-IL-10 is administered bycontinuous infusion so that an amount in the range of about 50 to 800 μgprotein is delivered per day (i.e., about 1 to 16 μg protein per kg ofbody weight per day PEG-IL-10). The daily infusion rate may be variedbased on monitoring of side effects and blood cell counts.

To prepare pharmaceutical compositions containing mono-PEG-IL-10, atherapeutically effective amount of PEG-IL-10 is admixed with apharmaceutically acceptable carrier or excipient. Preferably the carrieror excipient is inert. A pharmaceutical carrier can be any compatible,non-toxic substance suitable for delivering the IL-10 compositions ofthe invention to a patient. Examples of suitable carriers include normalsaline, Ringer's solution, dextrose solution, and Hank's solution.Non-aqueous carriers such as fixed oils and ethyl oleate may also beused. A preferred carrier is 5% dextrose/saline. The carrier may containminor amounts of additives such as substances that enhance isotonicityand chemical stability, e.g., buffers and preservatives, see, e.g.,Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: NationalFormulary, Mack Publishing Company, Easton, Pa. (1984). Formulations oftherapeutic and diagnostic agents may be prepared by mixing withphysiologically acceptable carriers, excipients, or stabilizers in theform of, e.g., lyophilized powders, slurries, aqueous solutions orsuspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's ThePharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;Gennaro (2000) Remington: The Science and Practice of Pharmacy,Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.)(1993) Pharmaceutical Dosage Forms: Parenteral Medications, MarcelDekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y.).

Compositions of the invention can be administered orally or injectedinto the body. Formulations for oral use can also include compounds tofurther protect the IL-10 from proteases in the gastrointestinal tract.Injections are usually intramuscular, subcutaneous, intradermal orintravenous. Alternatively, intra-articular injection or other routescould be used in appropriate circumstances.

When administered parenterally, pegylated IL-10 is preferably formulatedin a unit dosage injectable form (solution, suspension, emulsion) inassociation with a pharmaceutical carrier. See, e.g., Avis et al., eds.,Pharmaceutical Dosage Forms: Parenteral Medications, Dekker, N.Y.(1993); Lieberman et al., eds., Pharmaceutical Dosage Forms: Tablets,Dekker, N.Y. (1990); and Lieberman et al., eds., Pharmaceutical DosageForms: Disperse Systems, Dekker, N.Y. (1990). Alternatively,compositions of the invention may be introduced into a patient's body byimplantable or injectable drug delivery system, e.g., Urquhart et al.Ann. Rev. Pharmacol. Toxicol. 24:199-236, (1984); Lewis, ed., ControlledRelease of Pesticides and Pharmaceuticals Plenum Press, New York (1981);U.S. Pat. Nos. 3,773,919; 3,270,960; and the like. The pegylated IL-10can be administered in aqueous vehicles such as water, saline orbuffered vehicles with or without various additives and/or dilutingagents.

An effective amount for a particular patient may vary depending onfactors such as the condition being treated, the overall health of thepatient, the method route and dose of administration and the severity ofside affects (see, e.g., Maynard, et al. (1996) A Handbook of SOPs forGood Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001)Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

Typical veterinary, experimental, or research subjects include monkeys,dogs, cats, rats, mice, rabbits, guinea pigs, horses, and humans.

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced. Preferably, a biologic that will beused is derived from the same species as the animal targeted fortreatment, thereby minimizing a humoral response to the reagent. Methodsfor co-administration or treatment with a second therapeutic agent,e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, orradiation, are well known in the art (see, e.g., Hardman, et al. (eds.)(2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics,10^(th) ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.)(2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach,Lippincott, Williams & Wilkins, Phila., PA; Chabner and Longo (eds.)(2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams &Wilkins, Phila., PA). An effective amount of therapeutic will decreasethe symptoms, e.g., tumor size or inhibition of tumor growth, typicallyby at least 10%; usually by at least 20%; preferably at least about 30%;more preferably at least 40%, and most preferably by at least 50%.

VI. USES

The present invention provides methods of treating a proliferativecondition or disorder, e.g., cancer of the uterus, cervix, breast,prostate, testes, penis, gastrointestinal tract, e.g., esophagus,oropharynx, stomach, small or large intestines, colon, or rectum,kidney, renal cell, bladder, bone, bone marrow, skin, head or neck,skin, liver, gall bladder, heart, lung, pancreas, salivary gland,adrenal gland, thyroid, brain, e.g. gliomas, ganglia, central nervoussystem (CNS) and peripheral nervous system (PNS), and immune system,e.g., spleen or thymus. The present invention provides methods oftreating, e.g., immunogenic tumors, non-immunogenic tumors, dormanttumors, virus-induced cancers, e.g., epithelial cell cancers,endothelial cell cancers, squamous cell carcinomas, papillomavirus,adenocarcinomas, lymphomas, carcinomas, melanomas, leukemias, myelomas,sarcomas, teratocarcinomas, chemically-induced cancers, metastasis, andangiogenesis. The invention also contemplates reducing tolerance to atumor cell or cancer cell antigen, e.g., by modulating activity of aregulatory T cell (Treg) and or a CD8 T cell (see, e.g.,Ramirez-Montagut, et al. (2003) Oncogene 22:3180-3187; Sawaya, et al.(2003) New Engl. J. Med. 349:1501-1509; Farrar, et al. (1999) J.Immunol. 162:2842-2849; Le, et al. (2001) J. Immunol. 167:6765-6772;Cannistra and Niloff (1996) New Engl. J. Med. 334:1030-1038; Osborne(1998) New Engl. J. Med. 339:1609-1618; Lynch and Chapelle (2003) NewEngl. J. Med. 348:919-932; Enzinger and Mayer (2003) New Engl. J. Med.349:2241-2252; Forastiere, et al. (2001) New Engl. J. Med.345:1890-1900; Izbicki, et al. (1997) New Engl. J. Med. 337:1188-1194;Holland, et al. (eds.) (1996) Cancer Medicine Encyclopedia of Cancer,4^(th) ed., Academic Press, San Diego, Calif.).

In some embodiments, the present invention provides methods for treatinga proliferative condition, cancer, tumor, or precancerous condition suchas a dysplasia, with PEG-IL-10 and at least one additional therapeuticor diagnostic agent. The additional therapeutic agent can be, e.g., acytokine or cytokine antagonist, such as IL-12, interferon-alpha, oranti-epidermal growth factor receptor, doxorubicin, epirubicin, ananti-folate, e.g., methotrexate or fluoruracil, irinotecan,cyclophosphamide, radiotherapy, hormone or anti-hormone therapy, e.g.,androgen, estrogen, anti-estrogen, flutamide, or diethylstilbestrol,surgery, tamoxifen, ifosfamide, mitolactol, an alkylating agent, e.g.,melphalan or cis-platin, etoposide, vinorelbine, vinblastine, vindesine,a glucocorticoid, a histamine receptor antagonist, an angiogenesisinhibitor, radiation, a radiation sensitizer, anthracycline, vincaalkaloid, taxane, e.g., paclitaxel and docetaxel, a cell cycleinhibitor, e.g., a cyclin-dependent kinase inhibitor, a monoclonalantibody against another tumor antigen, a complex of monoclonal antibodyand toxin, a T cell adjuvant, bone marrow transplant, or antigenpresenting cells, e.g., dendritic cell therapy. Vaccines can beprovided, e.g., as a soluble protein or as a nucleic acid encoding theprotein (see, e.g., Le, et al., supra; Greco and Zellefsky (eds.) (2000)Radiotherapy of Prostate Cancer, Harwood Academic, Amsterdam; Shapiroand Recht (2001) New Engl. J. Med. 344:1997-2008; Hortobagyi (1998) NewEngl. J. Med. 339:974-984; Catalona (1994) New Engl. J. Med.331:996-1004; Naylor and Hadden (2003) Int. Immunopharmacol.3:1205-1215; The Int. Adjuvant Lung Cancer Trial Collaborative Group(2004) New Engl. J. Med. 350:351-360; Slamon, et al. (2001) New Engl. J.Med. 344:783-792; Kudelka, et al. (1998) New Engl. J. Med. 338:991-992;van Netten, et al. (1996) New Engl. J. Med. 334:920-921).

Also provided are methods of treating extramedullary hematopoiesis (EMH)of cancer. EMH is described (see, e.g., Rao, et al. (2003) Leuk.Lymphoma 44:715-718; Lane, et al. (2002) J. Cutan. Pathol. 29:608-612).

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the inventionsto the specific embodiments.

All citations herein are incorporated herein by reference to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited bythe terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled; and the invention is notto be limited by the specific embodiments that have been presentedherein by way of example.

Examples I. General Methods

Standard methods in molecular biology are described (Maniatis, et al.(1982) Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Sambrook and Russell (2001)Molecular Cloning, 3^(rd) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Wu (1993) Recombinant DNA, Vol. 217, AcademicPress, San Diego, Calif.). Standard methods also appear in Ausubel, etal. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wileyand Sons, Inc. New York, N.Y., which describes cloning in bacterialcells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast(Vol. 2), glycoconjugates and protein expression (Vol. 3), andbioinformatics (Vol. 4).

Methods for protein purification including immunoprecipitation,chromatography, electrophoresis, centrifugation, and crystallization aredescribed (Coligan, et al. (2000) Current Protocols in Protein Science,Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis,chemical modification, post-translational modification, production offusion proteins, glycosylation of proteins are described (see, e.g.,Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2,John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) CurrentProtocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY,NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for LifeScience Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech(2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production,purification, and fragmentation of polyclonal and monoclonal antibodiesis described (Coligan, et al. (2001) Current Protcols in Immunology,Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999)Using Antibodies, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Harlow and Lane, supra). Standard techniques forcharacterizing ligand/receptor interactions are available (see, e.g.,Coligan, et al. (2001) Current Protcols in Immunology, Vol. 4, JohnWiley, Inc., New York). Methods for making PEG-IL-10 are described,e.g., in U.S. Pat. No. 7,052,686.

Methods for flow cytometry, including fluorescence activated cellsorting (FACS), are available (see, e.g., Owens, et al. (1994) FlowCytometry Principles for Clinical Laboratory Practice, John Wiley andSons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2^(nd) ed.;Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry, JohnWiley and Sons, Hoboken, N.J.). Fluorescent reagents suitable formodifying nucleic acids, including nucleic acid primers and probes,polypeptides, and antibodies, for use, e.g., as diagnostic reagents, areavailable (Molecular Probes (2003) Catalogue, Molecular Probes, Inc.,Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

Standard methods of histology of the immune system are described (see,e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology andPathology, Springer Verlag, New York, N.Y.; Hiatt, et al. (2000) ColorAtlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.;Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, NewYork, N.Y.).

Methods for the treatment and diagnosis of cancer are described (see,e.g., Alison (ed.) (2001) The Cancer Handbook, Grove's Dictionaries,Inc., St. Louis, Mo.; Oldham (ed.) (1998) Principles of CancerBiotherapy, 3^(rd). ed., Kluwer Academic Publ., Hingham, Mass.;Thompson, et al. (eds.) (2001) Textbook of Melanoma, Martin Dunitz,Ltd., London, UK; Devita, et al. (eds.) (2001) Cancer: Principles andPractice of Oncology, 6^(th) ed., Lippincott, Phila, Pa.; Holland, etal. (eds.) (2000) Holland-Frei Cancer Medicine, BC Decker, Phila., PA;Garrett and Sell (eds.) (1995) Cellular Cancer Markers, Humana Press,Totowa, N.J.; MacKie (1996) Skin Cancer, 2^(nd) ed., Mosby, St. Louis;Moertel (1994) New Engl. J. Med. 330:1136-1142; Engleman (2003) Semin.Oncol. 30(3 Suppl. 8):23-29; Mohr, et al. (2003) Onkologie 26:227-233).

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains,glycosylation sites, and sequence alignments, are available (see, e.g.,GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, Md.); GCG WisconsinPackage (Accelrys, Inc., San Diego, Calif.); DeCypher® (TimeLogic Corp.,Crystal Bay, Nev.); Menne, et al. (2000) Bioinformatics 16: 741-742;Menne, et al. (2000) Bioinformatics Applications Note 16:741-742; Wren,et al. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res.14:4683-4690).

II. Pegylated IL-10

IL-10 (e.g., rodent or primate) was dialyzed against 50 mM sodiumphosphate, 100 mM sodium chloride pH ranges 5-7.4. A 1:1-1:7 molar ratioof 5K PEG-propyladehyde was reacted with IL-10 at a concentration of1-12 mg/ml in the presence of 0.75-30 mM sodium cyanoborohydride.Alternatively the reaction can be activated with picoline borane in asimilar manner. The reaction was incubated at 5-30° C. for 3-24 hours.

In particular, the pH of the pegylation reaction was adjusted to 6.3,7.5 mg/ml of hIL-10 was reacted with PEG to make the ratio of IL-10 toPEG linker 1:3.5. The final concentration of cyanoborohydride was 25 mM,and the reaction was carried out at 15° C. for 12-15 hours. FIG. 1 showsthe reaction kinetics of mono- and di-PEG-IL-10. The mono- and di-PEGIL-10 are the largest products of the reaction, with the concentrationof each at 50% at termination.

The reaction was quenched using an amino acid such as glycine and lysineor, alternatively, Tris buffers. Multiple purification methods have beenemployed such as gel filtration, anion and cation exchangechromatographies, and size exclusion to isolate the desired PEGylatedprototypes.

Alternatively, IL-10 is dialiyzed against 10 mM sodium phosphate pH 7.0,100 mM NaCl. The dialyzed IL-10 was diluted 3.2 times to a concentrationof about 0.5 to 12 mg/ml using the dialysis buffer. Prior to theaddition of the linker, SC-PEG-12K (Delmar Scientific Laboratories,Maywood, Ill.), 1 volume of 100 mM Na-tetraborate at pH 9.1 is addedinto 9 volumes of the diluted IL-10 to raise the pH of the IL-10solution to 8.6. The SC-PEG-12K linker is dissolved in the dialysisbuffer and the appropriate volume of the linker solution (1.8 to 3.6mole linker per mole of IL-10) is added into the diluted IL-10 solutionto start the pegylation reaction. The reaction is carried out at 5° C.in order to control the rate of the reaction. The reaction solution ismildly agitated during the pegylation reaction. When the mono-PEG-IL-10yield as determined by size exclusion HPLC (SE-HPLC), is close to 40%,the reaction was stopped by adding 1M glycine solution to a finalconcentration of 30 mM. The pH of the reaction solution is slowlyadjusted to 7.0 using an HCl solution and the reaction is 0.2 micronfiltered and stored at −80° C.

III. Efficacy Comparisons

To compare various prototypes of PEG-IL-10, 10 mice per treatment groupwere implanted subcutaneously with 10⁶ PDV6 squamous cell carcinomas inmatrigel, into the right flank, in 100 μL volume. Once the mean tumorsize reached 100 mm³ (approximately 2-3 weeks from implantation),treatment with the following murine PEG-IL-10 prototypes or control wasstarted, once a day (unless specified, the linker was PPA):

Control HEPES buffer 0.2 mpk 2 x 5K diPEG-IL-10 0.02 mpk 2 x 5KdiPEG-IL-10 0.2 mpk 1 x 5K monoPEG-IL-10 0.02 mpk 1 x 5K monoPEG-IL-100.2 mpk 1 x 5K monoPEG-IL-10 + 2 x 5K diPEG-IL-10 0.02 mpk 1 x 5KmonoPEG-IL-10 + 2 x 5K diPEG-IL-10 0.2 mpk 1 x 12K monoPEG-IL-10 (SClinker) 0.02 mpk 1 x 12K monoPEG-IL-10 (SC linker)The measured endpoints included tumor size (measured 2×/week), weights(measured 1×/week), and serum concentrations (measured at the start,midpoint and end of treatment). FIG. 2 shows the efficacies of thevarious prototypes described above.

All citations herein are incorporated herein by reference to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited bythe terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled; and the invention is notto be limited by the specific embodiments that have been presentedherein by way of example.

What is claimed is:
 1. A method of producing a mixture of mono- anddi-pegylated IL-10, wherein at least one PEG molecule is covalentlyattached to at least one amino acid residue of at least one subunit ofIL-10, comprising: a) reacting 1 mg/ml to 12 mg/ml of IL-10 protein withan activated PEG-linker such that the IL-10 to PEG-linker ratio is 1:1to 1:7.7, in the presence of 0.75 mM to 35 mM of a reducing agent, at apH of about 5.0 to 7.4 and a temperature of 5° C. to 30° C., for 12-15hours; and b) purifying the mixture of mono- and di-pegylated IL-10. 2.The method of claim 1, wherein the PEG-linker is selected from the groupconsisting of succinimidylcarbonate-PEG, PEG-butyraldehyde,PEG-pentaldehyde, PEG-amido-propionaldehyde,PEG-urethano-propioaldehyde, and PEG-propylaldehyde.
 3. The method ofclaim 2, wherein the PEG-linker is PEG-propylaldehyde.
 4. The method ofclaim 1, wherein the molecular mass of PEG comprising the PEG-linker isfrom 5,000 daltons to 20,000 daltons.
 5. The method of claim 4, whereinthe molecular mass of PEG comprising the PEG linker is 5,000 daltons. 6.The method of claim 1, wherein the reducing agent is selected from thegroup consisting of borohydride, sodium cyanoborohydride, amine borane,and picoline borane.
 7. The method of claim 6, wherein reducing agent isselected from the group consisting of sodium cyanoborohydride andpicoline borane.
 8. The method of claim 1, wherein the mixture of mono-and di-PEG is purified by chromatography selected from the groupconsisting of cation exchange, anion exchange, size exclusion, andhydrophobic interaction.
 9. The method of claim 9, wherein the mixtureof mono- and di-PEG-IL-10 is purified by size exclusion chromatography.10. A pharmaceutical composition comprising the mono- and di-PEG-IL-10produced by the method of claim 1 and a pharmaceutically acceptablecarrier.
 11. A method of producing a mixture of mono- and di-pegylatedIL-10, wherein at least one PEG molecule is covalently attached to atleast one amino acid residue of at least one subunit of IL-10,comprising a) reacting 7.5 mg/ml of IL-10 with an activated PEG-linkersuch that the IL-10 to PEG-linker ratio is 1:3.5, in the presence of 25mM of a reducing agent, at a pH of 6.3 and a temperature of 15° C., for15 hours; and b) purifying the mixture of mono- and di-pegylated IL-10.12. The method of claim 11, wherein the PEG-linker is selected from thegroup consisting of succinimidylcarbonate-PEG, PEG-butyraldehyde,PEG-pentaldehyde, PEG-amido-propionaldehyde,PEG-urethano-propioaldehyde, and PEG-propylaldehyde.
 13. The method ofclaim 12, wherein the PEG-linker is PEG-propylaldehyde.
 14. The methodof claim 11, wherein the molecular mass of PEG comprising the PEG-linkeris from 5,000 daltons to 20,000 daltons.
 15. The method of claim 14,wherein the molecular mass of PEG comprising the PEG linker is 5,000daltons.
 16. The method of claim 11, wherein the reducing agent isselected from the group consisting of borohydride, sodiumcyanoborohydride, amine borane, and picoline borane.
 17. The method ofclaim 16, wherein reducing agent is selected from the group consistingof sodium cyanoborohydride and picoline borane.
 18. The method of claim11, wherein the mixture of mono- and di-PEG is purified bychromatography selected from the group consisting of cation exchange,anion exchange, size exclusion, and hydrophobic interaction.
 19. Themethod of claim 18, wherein the mixture of mono- and di-PEG is purifiedby size exclusion chromatography.
 20. A pharmaceutical compositioncomprising the mono- and di-PEG-IL-10 produced by the method of claim 10and a pharmaceutically acceptable carrier.